Methods of treating castration-resistant prostate cancer with glucocorticoid receptor antagonists and anti-androgen receptor therapy

ABSTRACT

Embodiments of the invention are directed to the treatment of subjects with prostate cancer, in particular those with castration resistant prostate cancer, with glucocorticoid receptor antagonists. The prostate cancer may be one that has become resistant to androgen deprivation therapy, for example, by increase in glucocorticoid receptor expression and/or activity.

BACKGROUND OF THE INVENTION

The present application is a Continuation of U.S. application Ser. No.16/374,157, filed Apr. 3, 2019, which is a Continuation of U.S.application Ser. No. 15/704,726, filed Sep. 14, 2017 (now U.S. Pat. No.10,300,076, issued May 28, 2019), which is a Continuation of U.S.application Ser. No. 15/013,660, filed Feb. 2, 2016 (now U.S. Pat. No.9,801,893, issued Oct. 31, 2017), which is a Continuation of U.S.application Ser. No. 14/380,606, filed Aug. 22, 2014 (now U.S. Pat. No.9,289,436, issued Mar. 22, 2016), which is a U.S. national stageapplication under 35 U.S.C. § 371 of International Application No.PCT/US2013/027150, filed Feb. 21, 2013, which claims the benefit of, andpriority to, U.S. Provisional Patent Application Ser. No. 61/603,137,filed Feb. 24, 2012, the disclosures of which are hereby incorporated byreference in their entireties.

This invention was made with government support awarded by theDepartment of Defense and the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

Embodiments of this invention are directed generally to biology andmedicine. In certain aspects, methods and compositions for treating aprostate cancer patient with a glucocorticoid receptor (GR) antagonistare provided. More specifically, the methods comprise treating a subjectwith castration-resistant prostate cancer with a GR antagonist, inparticular in a subject that has previously received and demonstratedprostate cancer progression despite androgen-deprivation therapy.

BACKGROUND

Localized prostate cancers are treated with curative intent by surgeryor radiation, however, as many as 40% of patients will develop recurrentdisease over time, and it remains the second leading cause of cancerrelated death in men (Jemal et al., 2010; Ward and Moul, 2005). Thereare established predictors of prostate cancer recurrence or progressionand widely used prognostic nomograms, which in large part utilize commonpathologic criteria (Han et al., 2003; Pound et al., 1999; Makarov etal., 2007; Stephenson et al., 2006). Furthermore, there are multiplebiomarkers in development to help further hone prostate cancerprognostication (Fradet, 2009; Fiorentino et al., 2010). Nonetheless,the biologic factors modulating prostate cancer biology and progressiondespite anti-androgen therapy remain an important area of research.

The majority of prostate cancers rely on the androgen receptor (AR) forcell survival and proliferation, and the pathway remains important inthe progression of prostate cancer even in patients whose diseaseprogresses despite androgen deprivation therapy (Zegarra-Moro et al.,2002; Scher and Sawyers, 2005). Recently, serum/glucocorticoid-regulatedkinase 1 (SGK1) was found to be upregulated by AR activation in prostatecancer cell lines resulting in enhanced prostate cancer cell survival invitro (Shanmugam et al., 2007; Zou et al., 2009; Bolton et al., 2007).SGK1 is a serine/threonine protein kinase with 54% homology in itscatalytic domain to Akt and is involved in a multitude of metabolic andcell survival functions (Tessier and Woodgett, Jr., 2006). SGK1 istranscriptionally induced and its protein product plays an importantrole in cellular responses to stressors such as oxidation, heat, andultraviolet radiation (Tessier and Woodgett, 2006). SGK1 has been shownto be overexpressed in a proportion of human breast cancers (Sahoo etal., 2005) and to be important in protection from stress-inducedapoptosis in breast cancer cell lines (Mikosz et al., 2001; Wu et al.,2004). Similarly, androgen-sensitive prostate cancer cell lines thatectopically express SGK1 demonstrate increased survival followingandrogen-deprivation compared to those that do not overexpress the SGK1(Shanmugam et al., 2007). In addition, in vitro studies using smallinterfering RNAs targeting SGK1 or small molecule pharmacologicinhibitors of SGK1 demonstrate that inhibition of SGK1 activity leads todecreased androgen-mediated prostate cancer cell growth (Shanmugam etal., 2007; Sherk et al., 2008). The inventors showed SGK1 expression ishigh in primary prostate cancers and reduced following anti-androgentherapy (Szmulewitz et al., 2010) To the inventors' knowledge, there hasbeen only one other study examining SGK1 expression in primary humanprostate tumors; somewhat surprisingly, SGK1 expression wassignificantly decreased in prostate cancers as compared to prostatichypertrophy (Rauhala et al., 2005). In addition to the AR, SGK1 is alsoa direct target of glucocorticoid receptor (GR) activation in epithelialcells. Interestingly, standard chemotherapy regimens forcastrate-resistant prostate cancer include glucocorticoids such asprednisone, a GR agonist (Tannock et al., 2004; Petrylak et al., 2006).Although a proportion of patients show responses by a reduction in thetumor marker PSA and obtain palliative benefits from glucocorticoidtreatment, there are no phase III data demonstrating thatglucocorticoids provide a survival benefit (Fakih et al., 2002).Therefore, it is unclear how glucocorticoids function in metastaticprostate cancer. To date, there are a few reports examining GRexpression in human prostate cancer (Yemelyanov et al., 2007; Mohler etal., 1996) and no information on GR status in prostate cancer fromandrogen-deprived patients.

SUMMARY OF THE INVENTION

Embodiments concern methods, compositions, and apparati related toassessing, prognosing, and/or treating prostate cancer patients. Inparticular, it concerns using glucocorticoid receptor (GR) antagoniststo treat patients with prostate cancer, in particular those withmetastatic and castration resistant prostate cancer.

Some embodiments include generating an expression profile forglucocorticoid receptor (GR), which means obtaining the level ofexpression of AR or GR directly or indirectly by measuring or assayingactivity or expression. Methods include directly measuring or assayingthe level of expression or activity which refers to measuring orassaying a sample to determine the level of AR or GR expression (proteinor transcript) in the cell. Indirectly obtaining the level of expressionincludes measuring or assaying expression or activity of a gene orprotein that correlates with AR or GR expression or activity. In someembodiments, the level of AR or GR expression can be indirectly obtainedby measuring or assaying expression of a AR- or GR-responsive gene,which refers to a gene whose expression is affected in a dose-dependentmanner by AR or GR expression or activity. Expression refers to eitherprotein expression or RNA (transcript) expression. Methods may involveeither type of expression and a variety of assays are well known tothose of skill in the art. For example, quantitative PCR may beperformed to obtain RNA expression levels. The Affymetrix chip used inthe Examples also provides information regarding RNA expression levels.Alternatively, reagents to detect protein expression levels may beemployed in embodiments. Methods may involve probes, primers, and/orantibodies that are specific to GR or AR in order to assess expressionlevels.

In some embodiments, the activity level of GR is measured by assayingthe level of GR expression. In additional embodiments, GR expression isGR transcript expression. In other embodiments, GR expression is GRprotein expression. As discussed above, in some embodiments, theactivity level of GR is measured by assaying the expression level of oneor more GR-responsive genes. A GR-responsive gene may be one or more ofthe following: MCL1, SAP30, DUSP1, SGK1, SMARCA2, PTGDS, TNFRSF9, SFN,LAPTM5, GPSM2, SORT1, DPT, NRP1, ACSL5, BIRC3, NNMT, IGFBP6, PLXNC1,SLC46A3, C14orf139, PIAS1, IDH2, SERPINF1, ERBB2, PECAM1, LBH, ST3GAL5,IL1R1, BIN1, WIPF1, TFPI, FN1, FAM134A, NRIP1, RAC2, SPP1, PHF15,BTN3A2, SESN1, MAP3K5, DPYSL2, SEMA4D, STOM, or MAOA.

In some embodiments, there is a step of assaying or measuring theactivity level of glucocorticoid receptor (GR) in a biological samplefrom the patient containing prostate cancer cells. As discussed above,the activity level of GR can be obtained directly or indirectly. It isspecifically contemplated that levels of glucocorticoid activity orexpression refers to activity or expression of the genes or proteinsGRα, GRβ, or both. Unless specifically stated otherwise, the terms“glucocorticoid receptor” or “GR” refer to both forms. Embodimentsdiscussed with respect to glucocorticoid receptor or GR may also beimplemented solely with GRα or solely with GRβ.

Methods may also include obtaining a level of androgen receptor (AR)expression in prostate cancer cells from the patient. The level can beobtained by obtaining the results of an assay that measured the level ofAR expression. In some embodiments, the level is obtained by measuringor assaying the level of AR expression.

In one aspect of the invention, there is provided a method of treatingcastration-resistant prostate cancer in a subject comprisingadministering to said subject a glucocorticoid receptor (GR) antagonist.The subject may exhibit androgen-deprived prostate cancer, and/or maypreviously have been or is currently being treated with androgendeprivation therapy, such as with leuprolide goserelin, triptorelin,histrelin, degerelix or surgical castration. The subject may previouslyhave been or is currently being treated with an androgen receptor (AR)antagonist, such as MDV3100, ARN-509, flutamide, bicalutamide,nilutamide, or cyproterone acetate. The subject may exhibits prostatecancer with elevated GR expression. The GR antagonist may bebeclometasone, betamethasone, budesonide, ciclesonide, flunisolide,fluticasone, GSK650394, mifepristone, mometasone, or triamcinoclone. Infurther embodiments it may be CORT 0113083 or CORT 00112716.

The subject may be treated with a second prostate cancer therapy, suchas androgen deprivation therapy, including leuprolide goserelin,triptorelin, histrelin, or surgical castration. The second prostatecancer therapy may be an androgen synthesis inhibitor, such asketococonazole, abiraterone, TAK-700 and TOK001. The second prostatecancer therapy may be conventional chemotherapy, radiotherapy,cryotherapy, immunotherapy or surgery. The second prostate cancertherapy may be given prior to said GR antagonist, after said GRantagonist, or at the same time as said GR antagonist. The GR antagonistmay administered systemically, regionally or locally to a tumor site, ormay be administered orally, intravenously, intraarterially, into tumorvasculature or intratumorally. The GR antagonist is administered morethan once, such as 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 20, 25or more times.

The method may further comprising assessing GR expression in a prostatecancer tissue of said subject. The method may further comprisingassessing androgen receptor expression in a prostate cancer tissue ofsaid subject. The prostate cancer may be metastatic or recurrent. Thesubject may have previously been treated with both androgen-deprivationtherapy and an androgen receptor antagonist.

In another embodiment, there is provided a method of inhibiting theprogression of castration resistant prostate cancer in a subjectcomprising administering to said subject with a glucocorticoid receptor(GR) antagonist. A further embodiment comprises a method of preventingdevelopment of castration resistant prostate cancer in a subjectcomprising administering to said subject with a glucocorticoid receptor(GR) antagonist. Yet another embodiment comprise s method of inhibitingglucocorticoid receptor activity in prostate cancer tissues in a subjectcomprising administering to said subject with a GR antagonist. Anadditional embodiment comprises a method of treating castrationresistant prostate cancer in a subject comprising administering to saidsubject with a glucocorticoid receptor (GR) antagonist and a steroid.

Another embodiment comprises a method of treating prostate cancer in asubject comprising co-administering to said subject an androgen receptor(AR) antagonist and a glucocorticoid receptor (GR) antagonist. Further,an embodiment may comprise a method of treating prostate cancer in asubject whose tumor become resistant to anti-AR therapy comprising add aglucocorticoid receptor (GR) antagonist to their therapy. Still yet anadditional embodiment comprises a method of treating prostate cancer ina subject whose tumor become resistant to anti-AR therapy comprisingco-administering to said subject a glucocorticoid receptor (GR)antagonist and a cytotoxic chemotherapy. A further embodiment involves amethod of treating prostate cancer in a subject comprisingco-administering to said subject a glucocorticoid receptor (GR)antagonist and a drug that decreases circulating levels of testosterone.Another embodiment comprises a method of treating prostate cancer in asubject comprising alternating administrations to said subject of anandrogen receptor (AR) antagonist and a glucocorticoid receptor (GR)antagonist.

Embodiments may also include where the patient is treated with more thanone type of cancer therapy. This may be after the patient is determinedto have a particular prognosis or after the status of the patient's GRand AR expression profile is known. In some embodiments, a patient istreated with at least two of the following: radiation, chemotherapy, ora biologic. In particular embodiments, the patient may be treated withan anti-androgen, kinase inhibitor and/or anti-angiogenic agent.

In another embodiment, the method may comprises alternating ARantagonism and GR antagonism. The methods may therefore address tumorshift back and forth between signaling mechanisms that overcome receptorblockade. For example, the methods may include AR antagonist therapyfollowed by GR antagonist therapy, following by AR antagonist therapy,followed by GR antagonist therapy, and so on.

Any method may also include treating the patient for prostate cancer,which may include directly administering or providing a cancer therapy.In some embodiments, a practitioner or doctor may prescribe a cancertherapy that the patient administers to herself.

To achieve these methods, a doctor, medical practitioner, or their staffmay retrieve a biological sample from a patient for evaluation. Thesample may be a biopsy, such as a prostate tissue or tumor biopsy. Thesample may be analyzed by the practitioner or their staff, or it may besent to an outside or independent laboratory. The medical practitionermay be cognizant of whether the test is providing information regardingthe patient's level of GR and/or AR expression or activity, or themedical practitioner may be aware only that the test indicates directlyor indirectly that the test reflects that the patient has a particularprognosis or can be given a particular prognosis score. Furthermore, thepractitioner may know the patient's AR or GR status, such as AR+ or AR−,or GR+ or GR−. Alternatively, he or she may be aware only that the testor assay indicates the patient has a poor prognosis, or the worstprognosis.

Other embodiments include a computer readable medium having softwaremodules for performing a method comprising the acts of: (a) comparingglucocorticoid receptor data obtained from a patient's prostate cancersample with a reference; and (b) providing an assessment ofglucocorticoid receptor status to a physician for use in determining anappropriate therapeutic regimen for a patient. In further embodiments,the computer readable medium further comprises a software module forassessing estrogen receptor status of the patient's prostate cancersample.

Computer systems are also included. In some embodiments, they have aprocessor, memory, external data storage, input/output mechanisms, adisplay, for assessing glucocorticoid receptor activity, comprising: (a)a database; (b) logic mechanisms in the computer generating for thedatabase a GR-responsive gene expression reference; and (c) a comparingmechanism in the computer for comparing the GR-responsive geneexpression reference to expression data from a patient sample using acomparison model to determine a GR gene expression profile of thesample.

Other embodiments include an internet accessible portal for providingbiological information constructed and arranged to execute acomputer-implemented method for providing: (a) a comparison of geneexpression data of one or more GR-responsive genes in a patient samplewith a calculated reporter index; and (b) providing an assessment of GRactivity or expression to a physician for use in determining anappropriate therapeutic regime for a patient.

In addition to compiling, collecting and or processing data related toGR status, methods, media and systems may also include the sameembodiments with respect to data related to AR status. Such aspects maybe instead of or in addition to the aspects related to GR status ordata.

Embodiments also include methods of killing prostate cancer cellscomprising administering to a prostate cancer patient an effectiveamount of a combination of anti-cancer compounds, wherein the anticancercompounds comprise a glucocorticoid receptor antagonist and achemotherapeutic. In other embodiments, there are methods for treatingprostate cancer in a patient comprising administering to the patient aneffective amount of glucocorticoid receptor antagonist and achemotherapeutic. In further embodiments, methods are provided fortreating chemotherapy-insensitive prostate cancer cells comprisingadministering to a prostate cancer patient an effective amount of aglucocorticoid receptor antagonist followed by chemotherapy.

Other methods include methods for treating prostate cancer in a patientcomprising: a) administering radiation or at least a firstchemotherapeutic to the patient; b) subsequently administering aneffective amount of a glucocorticoid receptor antagonist to the patient;and, c) administering radiation again or at least a secondchemotherapeutic to the patient after the glucocorticoid receptorantagonist is administered to the patient.

In some embodiments, there are methods for treating prostate cancer in apatient comprising: a) administering an effective amount of aglucocorticoid receptor antagonist to the patient, wherein the patientexpresses detectable levels of GR prior to administration of the GRantagonist; b) then administering an effective amount of radiation or atleast one chemotherapeutic.

It is contemplated that in methods described herein, prostate cancercells may undergo apoptosis following treatment set forth herein.Moreover, in some embodiments, the combination of a glucocorticoidreceptor antagonist and an anticancer agent or compound induces moreapoptosis than treatment with just the anticancer treatment alone.

Glucocorticoid receptor antagonists are known to those of skill in theart. It refers to a compound or substance that that does not provoke abiological response itself upon binding to the glucocorticoid receptor,but blocks or dampens agonist-mediated responses. Examples include, butare not limited to, beclometasone, betamethasone, budesonide,ciclesonide, flunisolide, fluticasone, mifepristone, mometasone, andtriamcinolone. In further embodiments it may be CORT 0113083 or CORT00112716. In additional embodiments, the glucocorticoid receptorantagonist has undetectable level or a lower level of activity as aprogesterone receptor antagonist. In certain embodiments, theglucocorticoid receptor antagonist has greater than 10-fold, 50-fold,100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold lowerbinding activity (or any range derivable therein) for another hormonereceptor compared to its binding activity for glucocorticoid receptor.In specific embodiments the hormone receptor is estrogen receptor orprogesterone receptor.

In some embodiments, a patient had been previously treated with ananti-cancer therapy, such as radiation, chemotherapy, or immunotherapy(or a combination or multiple therapies thereof). In certainembodiments, a first anti-cancer therapy prior to therapy withglucocorticoid receptor antagonist was last administered more than twoweeks prior to the glucocorticoid receptor antagonist or its combinationwith a second anti-cancer therapy. In certain embodiments, this firstanti-cancer therapy that does not include a glucocorticoid receptorantagonist was last administered to the prostate cancer patient at least7, 8, 9, 10, 11, 12, 13, 14 days, and/or 1, 2, 3, 4, or 5 weeks, and/or1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months prior to treatment witha glucocorticoid receptor antagonist. Treatment methods may be appliedto prostate cancer or prostate cancer cells that are chemo-resistant orprostate cancer cells that are not chemo-sensitive. Moreover, treatmentmay be applied to prostate cancer or to prostate cancer cells that werepreviously administered a first apoptosis inducing agent, but wereresistant to apoptosis.

Methods involve treating prostate cancer, particularly an ARantagonist-resistant prostate cancer, with a combination of therapiesthat includes a glucocorticoid receptor antagonist and an anticancertherapy that induces apoptosis (together they may be referred to as acombination of anti-cancer agents or compounds), such as achemotherapeutic or an anti-androgen receptor (AR) antagonist such asMDV-3100. In some embodiments, the chemotherapeutic is capecitabine,carboplatin, cyclophosphamide (Cytoxan), daunorubicin, docetaxel(Taxotere), doxorubicin (Adriamycin), epirubicin (Ellence), fluorouracil(also called 5-fluorouracil or 5-FU), gemcitabine, eribulin,ixabepilone, methotrexate, mitomycin C, mitoxantrone, paclitaxel(Taxol), thiotepa, vincristine, or vinorelbine, or a combination ofthese agents. In other embodiments, therapy with a glucocorticoidreceptor antagonist is combined MDV-3100, abiraterone, radiation,chemotherapeutic(s) and radiation, a combination of chemotherapeutics,or a combination of one or more chemotherapeutic agents and ananti-androgen or anti-androgen receptor drug.

It is contemplated that in some embodiments of the combination therapythe glucocorticoid receptor antagonist is administered within 5, 10, 30,45, 60 minutes, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and/or 1, 2, 3, 4, 5, 6, 7days, or any combination thereof within administration of at least oneor the combination of the anti-cancer agents or compounds. In specificembodiments, the glucocorticoid receptor antagonist is administeredwithin 2 hours, 12 hours or 24 hours of administration of an anticanceragent or compound (or a combination of such agents or compounds).

In a further embodiment, the glucocorticoid receptor (GR) antagonists ofthe present invention can be combined with anti-kinases such asmammalian target of rapamycin (mTOR) inhibitors. Non-limiting examplesof mTOR inhibitors include rapamycin, epigallocatechin gallate (EGCG),caffeine curcumin, resveratrol, temsirolimus, everolimus, andridaforolimus, and derivatives and analogues thereof. This combinationcan be used in compositions and methods disclosed throughout thespecification.

It is specifically contemplated that treatment may continue or berepeated. In some embodiments, once treated with the combination of aglucocorticoid receptor antagonist and at least one anticancer agent orcompound, all or part of the treatment may be repeated alone or incombination with a different anticancer agent or compound.

In certain embodiments, the glucocorticoid receptor antagonist isadministered prior to as the other agent or therapy included in thecombination therapy. In certain embodiments, the glucocorticoid receptorantagonist is administered 5, 10, 30, 45, 60 minutes, and/or 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 hours, and/or 1, 2, 3, 4, 5, 6, 7 days, or any combination thereofprior to administration of at least one or the combination of theanti-cancer agents or compounds. It is specifically contemplated that insome embodiments, the glucocorticoid receptor antagonist is given priorto administration of the anticancer agent or compound but that theglucocorticoid receptor antagonist is also given concurrently with orafter administration of the initial or a subsequent dose of theanticancer agent or compound. As discussed throughout, the anticanceragent or compound may be in a combination of such agents or compounds.In certain embodiments, the glucocorticoid receptor antagonist isadministered up to three days prior to administering the anticanceragent or compound.

Additionally or alternatively, the glucocorticoid receptor antagonist isadministered after administration of the other agent or therapy includedin the combination therapy. In certain embodiments, the glucocorticoidreceptor antagonist is administered 5, 10, 30, 45, 60 minutes, and/or 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24 hours, and/or 1, 2, 3, 4, 5, 6, 7 days, or any combinationthereof after administration of at least one or the combination of theanti-cancer agents or compounds. It is specifically contemplated that insome embodiments, the glucocorticoid receptor antagonist is given afterto administration of the anticancer agent or compound; suchadministration may be repeated. As discussed throughout, the anticanceragent or compound may be in a combination of such agents or compounds.In certain embodiments, the glucocorticoid receptor antagonist isadministered up to three days after administering the anticancer agentor compound.

Compositions are contemplated to include a glucocorticoid receptorantagonist and any other anticancer compound discussed herein. In someembodiments, the composition is in a pharmaceutically acceptableformulation.

Use of the one or more compositions may be employed based on methodsdescribed herein. Other embodiments are discussed throughout thisapplication. Any embodiment discussed with respect to one aspect of theinvention applies to other aspects of the invention as well and viceversa. The embodiments in the Example section are understood to beembodiments that are applicable to all aspects of the technologydescribed herein.

“Cancer prognosis” generally refers to a forecast or prediction of theprobable course or outcome of the cancer. As used herein, cancerprognosis includes the forecast or prediction of any one or more of thefollowing: duration of survival of a patient susceptible to or diagnosedwith a cancer, duration of recurrence-free survival, duration ofprogression free survival of a patient susceptible to or diagnosed witha cancer, response rate in a group of patients susceptible to ordiagnosed with a cancer, and/or duration of response in a patient or agroup of patients susceptible to or diagnosed with a cancer.

In certain aspects, prognosis is an estimation of the likelihood ofmetastasis free survival of said patient over a predetermined period oftime, e.g., over a period of 5 years.

In further aspects, prognosis is an estimation of the likelihood ofdeath of disease of said patient over a predetermined period of time,e.g., over a period of 5 years.

The term “recurrence” refers to the detection of prostate cancer in formof metastatic spread of tumor cells, local recurrence, contralateralrecurrence or recurrence of prostate cancer at any site of the body ofthe patient after prostate cancer had been substantially undetectable orresponsive to treatments.

As used herein, “treatment” or “therapy” is an approach for obtainingbeneficial or desired clinical results. This includes: reduce the numberof cancer cells; reduce the tumor size; inhibit (i.e., slow to someextent and/or stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and/or stop) tumor metastasis;inhibit, to some extent, tumor growth; and/or relieve to some extent oneor more of the symptoms associated with the disorder, shrinking the sizeof the tumor, decreasing symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, delaying theprogression of the disease, and/or prolonging survival of patients.

The term “therapeutically effective amount” refers to an amount of thedrug that may reduce the number of cancer cells; reduce the tumor size;inhibit (i.e., slow to some extent and preferably stop) cancer cellinfiltration into peripheral organs; inhibit (i.e., slow to some extentand preferably stop) tumor metastasis; inhibit, to some extent, tumorgrowth; and/or relieve to some extent one or more of the symptomsassociated with the disorder. To the extent the drug may prevent growthand/or kill existing cancer cells, it may be cytostatic and/orcytotoxic. For cancer therapy, efficacy in vivo can, for example, bemeasured by assessing the duration of survival, time to diseaseprogression (TTP), the response rates (RR), duration of response, and/orquality of life.

The terms “overexpress,” “overexpression,” “overexpressed,”“up-regulate,” or “up-regulated” interchangeably refer to a biomarkerthat is transcribed or translated at a detectably greater level, usuallyin a cancer cell, in comparison to a non-cancer cell or cancer cell thatis not associated with the worst or poorest prognosis. The term includesoverexpression due to transcription, post transcriptional processing,translation, post-translational processing, cellular localization,and/or RNA and protein stability, as compared to a non-cancer cell orcancer cell that is not associated with the worst or poorest prognosis.Overexpression can be detected using conventional techniques fordetecting mRNA (i.e., RT-PCR, PCR, hybridization) or proteins (i.e.,ELISA, immunohistochemical techniques, mass spectroscopy).Overexpression can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% ormore in comparison to a normal cell or cancer cell that is notassociated with the worst or poorest prognosis. In certain instances,overexpression is 1-fold, 2-fold, 3-fold, 4-fold 5, 6, 7, 8, 9, 10, or15-fold or more higher levels of transcription or translation incomparison to a non-cancer cell or cancer cell that is not associatedwith the worst or poorest prognosis.

“Biological sample” includes sections of tissues such as biopsy andautopsy samples, and frozen sections taken for histologic purposes. Suchsamples include prostate cancer tissues, cultured cells, e.g., primarycultures, explants, and transformed cells. A biological sample istypically obtained from a mammal, such as a primate, e.g., human.

A “biopsy” refers to the process of removing a tissue sample fordiagnostic or prognostic evaluation, and to the tissue specimen itselfAny biopsy technique known in the art can be applied to the diagnosticand prognostic methods of the present invention. The biopsy techniqueapplied will depend on the tissue type to be evaluated (e.g., prostate),the size and type of the tumor, among other factors. Representativebiopsy techniques include, but are not limited to, excisional biopsy,incisional biopsy, needle biopsy, and surgical biopsy. An “excisionalbiopsy” refers to the removal of an entire tumor mass with a smallmargin of normal tissue surrounding it. An “incisional biopsy” refers tothe removal of a wedge of tissue that includes a cross-sectionaldiameter of the tumor. A diagnosis or prognosis made by endoscopy orfluoroscopy can require a “core-needle biopsy,” or a “fine-needleaspiration biopsy” which generally obtains a suspension of cells fromwithin a target tissue. Biopsy techniques are discussed, for example, inHarrison's Principles of Internal Medicine (2005). Obtaining a biopsyincludes both direct and indirect methods, including obtaining thebiopsy from the patient or obtaining the biopsy sample after it isremoved from the patient.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” It is also contemplatedthat anything listed using the term “or” may also be specificallyexcluded.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-C. Immunohistochemical analysis of prostate cancer tissue.(FIG. 1A) SGK1 expression. Representative pictures showing SGK1expression is increased in prostate cancer cells compared to benignsurrounding epithelium (1^(st) panel-20× magnification, black line isdrawn to show demarcation of cancerous area). In TN-PC (middle twopanels) SGK1 expression is variable. In AD-PC, high SGK1 expression wasless frequent (40×). (FIG. 1B) GR was expressed in a higher percentageof AD-PC compared to TN-PC (40×). (FIG. 1C) AR expression wasuniversally positive and predominantly nuclear in both TN-PC and AD-PC(40×).

FIGS. 2A-C. PSA progression free survival estimates. Kaplan-Meierlog-rank survival estimates of progression-free survival for (FIG. 2A).All patients (FIG. 2B). Stratified by Gleason grade low (5-6) versusintermediate/high (7-9). (FIG. 2C) Stratified by SGK1 expression high(3+) versus low (0 to 2+).

DETAILED DESCRIPTION OF THE INVENTION

SGK1 has recently been identified as an AR-regulated target gene thatencodes a protein kinase important in prostate cancer cell survival.Currently, there is a paucity of knowledge regarding SGK1 expression andits clinical significance in primary prostate cancers. The inventorsexamined the expression of SGK1 along with its nuclear receptorregulators AR and GR in both untreated and androgen-deprived humanprostate cancer. They found that SGK1 is expressed in virtually allprostate cancers, but that the level of SGK1 expression is variable.SGK1 expression was consistently more intense in tumor epithelial cellscompared to unaffected surrounding prostate tissue, supporting thenotion that increased AR activity induces SGK1 expression. Thisobservation contradicts a previous study examining SGK1 expression,which demonstrated a decrease in expression in tumor tissue samples whencompared to benign prostatic hypertrophy (Rauhala et al., 2005). Thisdifference may be due to the comparison of cancer to benign prostatichypertrophy in the previous study, rather than a comparison tounaffected adjacent normal prostate tissue as in this study.Furthermore, the finding that SGK1 expression decreases followingandrogen-deprivation therapy supports the finding that SGK1 expressionis AR-mediated.

Although only statistically significant at 5 years, it is nonethelessinteresting that the inventors found an increased risk of prostatecancer recurrence in patients with lower SGK1 expression. This findingcontradicted the inventors' initial hypothesis that high SGK1 expressionin untreated primary prostate cancers would predict an increased risk ofrecurrence secondary to enhanced cancer cell survival. However, becauseSGK1 is an AR target gene, lower expression of SGK1 despite strong ARexpression may reflect aberrant androgen pathway signaling associatedwith a less differentiated tumor phenotype. This hypothesis isconsistent with the association between a higher Gleason grade and lowerSGK1 expression found in this study. In support of this hypothesis,another study examining the expression of the AR target gene PSA/HK3 inprostate cancer found a similar inverse correlation between thisAR-regulated gene and biochemical recurrence (Sterbis et al., 2008). Onthe other hand, a recent publication by Donovan et al. (2010) using anovel qualitative immunofluorescence scoring system for nuclear ARexpression found an association of higher nuclear AR expression withincreased prostate cancer specific mortality. Other studies examining AR(NR3C4) mRNA levels in primary tumor tissue also found an increase inbiochemical relapse in patients with higher AR (NR3C4) mRNA compared tobenign surrounding tissues (Rosner et al., 2007; Li et al., 2004). Thesefindings likely reflect a difference between measurable AR expressionand actual AR pathway activity reflecting the complexity of AR pathwaysignaling in prostate cancer biology. There are multiple possiblemechanisms underlying potentially decreased AR signaling, even in thesetting of intact testosterone. These are only now coming to light, andinclude AR splice variants and mutations (Guo et al., 2009; Koochekpour,2010; Sun et al., 2010).

It is clear that this study has several limitations. Foremost is thatthe percentage of tumors with low SGK1 expression was only ˜25% of thetotal sample size; analysis of this population is therefore limited. Thesecond major limitation is that many patients were lost to follow-upover time. The wide range of follow-up from 6 weeks to over 15 years mayconfound the PFS analyses. In addition, several of the statisticalassociations between SGK1 expression and clinical parameters weresuggestive of an association while not meeting the P≤0.05 cut-off,although the association with relapse at 5 years was statisticallysignificant. A larger sample size, potentially enriched for patientswith higher grade disease, would likely strengthen these findings.Furthermore, more consistent long-term follow-up would also potentiallyallow more robust statistical analyses to be made. Such studies,including multivariate analyses, receiver operating curves, andcorrelation coefficients would clearly be necessary to justify the useof SGK1 staining as a prognostic biomarker.

Although SGK1 is a known effector of the glucocorticoid pathway (Mikoszet al., 2001) and glucocorticoids are utilized in systemic therapy forcastrate-resistant prostate cancer, little is known about either GRexpression in prostate cancer or how glucocorticoids may be exerting atherapeutic effect. To the inventors' knowledge, there have only beentwo prior studies investigating GR expression in prostate cancer(Yemelyanov et al., 2007; Mohler et al., 1996). In line with previous GRexpression data, the inventors have also found that GR was expressed inapproximately a third of PC samples when compared to adjacent normalprostate tissue. Interestingly, this study demonstrates that GR isexpressed in a higher proportion of androgen-deprived compared totreatment-naïve primary prostate cancer samples, which neither of theprior studies examined. Furthermore, of the five castrate-resistantsamples from the AD-PC group, four overexpressed GR (80%). Although thesample size is small, this finding is interesting and could be explainedby the fact that in an androgen-depleted environment, GR expressionincreases to compensate for decreased AR activity. AR and GR sharesimilar DNA binding domain sequences as well as some of the samedownstream effector genes, including SGK1 (Cleutjens et al., 1997; Ho etal., 1993; Chen et al., 1997). It is well known that the AR remainsrelevant in the progression to castrate-resistant prostate cancer(Zegarra-Moro et al., 2002; Scher and Sawyers, 2005). The inventors alsoconsidered it possible that in an androgen-depleted, castrate-resistantenvironment, GR might retain a role in transcriptional regulation ofandrogen-regulated genes such as SGK1, and could serve as a survivalpathway for castrate-resistant prostate cancer. The inventors'observation that SGK1 expression remained high in nearly half ofandrogen-deprived cancers is consistent with this hypothesis.

Thus, in further studies, the inventors tested whether, followingandrogen-deprivation therapy (ADT) and the loss of AR activity inprostate cancer cells, administration of GR antagonists would have aneffect. As shown herein, the inventors demonstrated that in the contextof androgen receptor antagonism, the levels of glucocorticoid receptor(GR) within human castrate-resistant prostate cancer cell linesincreases compared to those who have not been treated with ARinhibition, GR activation makes these castrate-resistant cell lines moreresistant to AR inhibition, and that treatment of these cell lines withGR antagonists results in synergistic growth inhibition. These and otheraspects of the invention are described in detail below.

I. Prostate Cancer

Prostate cancer is a form of cancer that develops in the prostate, agland in the male reproductive system. Many prostate cancers are slowgrowing; however, it remains the leading cause of cancer death in men inthe United States. (en.wikipedia.org/wiki/Prostate_cancer—cite_note-0).The cancer cells may metastasize (spread) from the prostate to otherparts of the body, particularly the bones and lymph nodes. Prostatecancer may cause pain, difficulty in urinating, problems during sexualintercourse, or erectile dysfunction. Other symptoms can potentiallydevelop during later stages of the disease.

Rates of detection of prostate cancers vary widely across the world,with South and East Asia detecting less frequently than in Europe, andespecially the United States. Prostate cancer tends to develop in menover the age of fifty and although it is one of the most prevalent typesof cancer in men, many never have symptoms, undergo no therapy, andeventually die of other causes. This is because cancer of the prostateis, in most cases, slow-growing, symptom-free, and since men with thecondition are older they often die of causes unrelated to the prostatecancer, such as heart/circulatory disease, pneumonia, other unconnectedcancers, or old age. On the other hand, the more aggressive prostatecancers account for more cancer-related mortality than any other cancerexcept lung cancer. About two-thirds of cases are slow growing, theother third more aggressive and fast developing.

Many factors, including genetics and diet, have been implicated in thedevelopment of prostate cancer. The presence of prostate cancer may beindicated by symptoms, physical examination, prostate-specific antigen(PSA), or biopsy. The PSA test increases cancer detection but does notdecrease mortality(en.wikipedia.org/wiki/Prostate_cancer—cite_note-BMJ2010-4). Moreover,prostate test screening is controversial at the moment and may lead tounnecessary, even harmful, consequences in some patients. Nonetheless,suspected prostate cancer is typically confirmed by taking a biopsy ofthe prostate and examining it under a microscope. Further tests, such asCT scans and bone scans, may be performed to determine whether prostatecancer has spread.

Management strategies for prostate cancer should be guided by theseverity of the disease. Many low-risk tumors can be safely followedwith active surveillance. Curative treatment generally involves surgery,various forms of radiation therapy, or, less commonly, cryosurgery;hormonal therapy and chemotherapy are generally reserved for cases ofadvanced disease (although hormonal therapy may be given with radiationin some cases).

The age and underlying health of the man, the extent of metastasis,appearance under the microscope and response of the cancer to initialtreatment are important in determining the outcome of the disease. Thedecision whether or not to treat localized prostate cancer (a tumor thatis contained within the prostate) with curative intent is a patienttrade-off between the expected beneficial and harmful effects in termsof patient survival and quality of life.

Early prostate cancer usually causes no symptoms. Sometimes, however,prostate cancer does cause symptoms, often similar to those of otherprostate diseases such as benign prostatic hyperplasia. These includefrequent urination, nocturia (increased urination at night), difficultystarting and maintaining a steady stream of urine, hematuria (blood inthe urine), and dysuria (painful urination).

Prostate cancer is associated with urinary dysfunction as the prostategland surrounds the prostatic urethra. Changes within the gland,therefore, directly affect urinary function. Because the vas deferensdeposits seminal fluid into the prostatic urethra, and secretions fromthe prostate gland itself are included in semen content, prostate cancermay also cause problems with sexual function and performance, such asdifficulty achieving erection or painful ejaculation.

Advanced prostate cancer can spread to other parts of the body, possiblycausing additional symptoms. The most common symptom is bone pain, oftenin the vertebrae (bones of the spine), pelvis, or ribs. Spread of cancerinto other bones such as the femur is usually to the proximal part ofthe bone. Prostate cancer in the spine can also compress the spinalcord, causing leg weakness and urinary and fecal incontinence.

The only test that can fully confirm the diagnosis of prostate cancer isa biopsy, the removal of small pieces of the prostate for microscopicexamination. However, prior to a biopsy, less invasive testing can beconducted. There are several tests that can be used to gather moreinformation about the prostate and the urinary tract. Digital rectalexamination (DRE) may allow a doctor to detect prostate abnormalities.Cystoscopy shows the urinary tract from inside the bladder, using athin, flexible camera tube inserted down the urethra. Transrectalultrasonography creates a picture of the prostate using sound waves froma probe in the rectum. PSA level tests also are used frequently toscreen for higher risk patients.

Prostate cancer is initially “hormone dependent”, meaning its growth andprogression is dependent on androgen hormones. The majority of thesehormones are produced by the testicles. Most hormone dependent cancersbecome refractory after one to three years and resume growth despitehormone therapy. Previously considered “hormone-refractory prostatecancer” or “androgen-independent prostate cancer,” the term“castration-resistant” has replaced “hormone refractory” because whilethey are no longer responsive to castration treatment (reduction ofavailable androgen/testosterone/DHT by chemical or surgical means),these cancers still show reliance upon hormones for androgen receptoractivation. Before 2004, all treatments for castration-resistantprostate cancer (CRPC) were considered palliative and not shown toprolong survival. However, there are now several treatments available totreat CRPC that improve survival.

The cancer chemotherapeutic docetaxel has been used as treatment forCRPC with a median survival benefit of 2 to 3 months. Docetaxel's FDAapproval in 2004 was significant as it was the first treatment proven toprolong survival in CRPC. In 2010, the FDA approved a second-linechemotherapy treatment known as cabazitaxel. Off-label use of the oraldrug ketoconazole is sometimes used as a way to further manipulatehormones with a therapeutic effect in CRPC. However, many side effectsare possible with this drug and abiraterone is likely to supplant usagesince it has a similar mechanism of action with less toxic side effects.The immunotherapy treatment with sipuleucel-T is also effective in thetreatment of CRPC with a median survival benefit of 4.1 months.

The second line hormonal therapy abiraterone (Zytiga) completed a phase3 trial for CRPC patients who have failed chemotherapy in 2010. Resultswere positive with overall survival increased by 4.6 months whencompared to placebo. On Apr. 28, 2011, the U.S. Food and DrugAdministration approved abiraterone acetate in combination withprednisone to treat patients with late-stage (metastatic)castration-resistant prostate cancer patients who have received priordocetaxel (chemotherapy). Another anti-androgen pathway therapy,MDV3100, is an extremely potent and specific inhibitor of the androgenreceptor. A phase III clinical trial of MDV3100 in castration-resistantprostate cancer patients who have received prior docetaxel(chemotherapy) was reported in 2012, and similarly demonstrated a 4-5month survival advantage. It has yet to be approved by the FDA, howeverits approval is likely.

II. Glucocortocoid and Androgen Receptor Antagonists

A. Glucocortocoid Receptor Antagonists

Early research on steroidal ligands led to the identification of thenon-selective GR antagonist RU-486 (mifepristone) and the GR-selectivesteroid RU-43044. Others include dual antagonist-agonists beclometasone,betamethasone, budesonide, ciclesonide, flunisolide, fluticasone,mometasone, and triamcinolone. Structurally-related compounds that alsoare GR antagonists include octahydrophenanthrenes, spirocyclicdihydropyridines, triphenylmethanes and diaryl ethers, chromenes,dibenzyl anilines, dihydroisoquinolines, pyrimidinediones, azadecalins,aryl pyrazolo azadecalins, 11-monoaryl steroids, phenanthrenes, dibenzol[2.2.2]cycloctaines and derivatives, dibenzoclyclohepatnes and theirderivatives, dibenzyl anilinesulfonamides and their derivatives,dihetero(aryl) pentanol, chromene derivatives, Azadecalins, arylquinolones, 11,21-bisaryl steroids and 11-aryl, and 16-hydroxy steroids.Collectively, these compounds are referred to herein as “GRantagonists.” See Moeler et al., Expert Opin., 17 (1), 2007).

B. Androgen Receptor Antagonists

Cyproterone Acetate. Cyproterone acetate is a progestional anti-androgenthat directly inhibits the androgen receptor. Co-cyprindol combines(COCs) 2 mg CPA with 35 μg ethinyl estradiol and it has been suggestedthat the higher amount of estrogen in these agents carries a greaterpotential for VTE compared with conventional lower estrogen-containingCOCs. However, evidence for adverse effects of co-cyprindol concerninghigher VTE risk suggests it is no greater than with third-generationCOCs. Cyproterone is no longer in used in the United States.

Spironalactone. Spironalactone has inhibitory actions on both theandrogen receptor and 5α-reductase. Spironalactone is not without sideeffects, although these are largely dose dependent. Potentialhyperkalemia, fatigue, headache, fluid retention and, rarely, melasmahave been observed. Animal studies have reported an association withbreast carcinoma in rodents but this has not been replicated in humanstudies. All patients should undergo regular monitoring of theirelectrolytes owing to the potassium-retaining effects on the kidney.

Flutamide. Flutamide is a nonsteroidal potent androgen antagonist, mostroutinely used in the treatment of prostate cancer. In terms of safety,fatal hepatotoxicity has been reported with flutamide. Initial warningsof hepatotoxicity were reported from patients using doses of 750 mgdaily and reported prior to any dose-response studies being undertaken.The use of lower doses of flutamide is currently under investigation. Nocases of hepatic impairment with flutamide doses of 125 mg/day or lesshave been reported, and placebo-controlled data suggest that doses aslow as 250-375 mg/day may be effective in antagonizing androgenproduction in females, especially when combined with adrospirenone-containing contraceptive.

Bicalutamide. Bicalutamide (marketed as Casodex, Cosudex, Calutide,Kalumid) is an oral non-steroidal anti-androgen used in the treatment ofprostate cancer and hirsutism. It is recommended 50 mg once daily incombination with a luteinizing hormone-releasing hormone analogue orsurgical castration or upon progression after castration as a secondaryhormonal maneuver.

Nilutamide. Nilutamide is an antiandrogen medication used in thetreatment of advanced stage prostate cancer. Nilutamide blocks theandrogen receptor, preventing its interaction with testosterone. Becausemost prostate cancer cells rely on the stimulation of the androgenreceptor for growth and survival, nilutamide can prolong life in menwith prostate cancer. Nilutamide is marketed under the name Nilandron inthe United States and under the name Anandron in Canada. It is used incombination with a luteinizing hormone-releasing hormone analogue orsurgical castration or upon progression after castration as a secondaryhormonal maneuver.

MDV3100. MDV3100 is an experimental androgen receptor antagonist drugdeveloped by Medivation for the treatment of castration-resistantprostate cancer currently in Phase 3 clinical trials. Medivation hasreported up to an 89% decrease in prostate specific antigen serum levelsafter a month of taking the medicine. Early preclinical studies alsosuggest that MDV3100 inhibits breast cancer cell growth.

MDV3100 has approximately five-fold higher binding affinity for theandrogen receptor (AR) compared to the antiandrogen bicalutamide. Asopposed to bicalutamide, MDV3100 does not promote translocation of AR tothe nucleus and in addition prevents binding of AR to DNA and AR tocoactivator proteins. When LNCaP cells (a prostate cancer cell line)engineered to express elevated levels of AR (as found in patients withadvanced prostate cancer) were treated with MDV3100, the expression ofandrogen dependent genes PSA and TMPRSS2 were down regulated in contrastto bicalutamide where the expression was upregulated. In VCaP cellswhich over express androgen receptors, MDV3100 induced apoptosis,whereas bicalutamide did not. Furthermore MDV3100 behaves as anantagonist of the W741C mutant androgen receptor in contrast tobicalutamide which behaves as a pure agonist when bound to the W741Cmutant.

MDV 3100 was found clinically active for metastatic castration-resistantprostate cancer patients in ongoing phase I and II trials. PSA leveldecreased more than 50 percent in 40/65 chemo-naive patients and 38/75chemotherapy-treated patients. Recent long-term follow up data fromthese early clinical studies, announced in February 2011, were positive.Median time to radiographic progression was 56 weeks for chemo-naivepatients and 25 weeks for the post-chemotherapy population. Medivationis conducting several international phase III trials. The first trial,known as AFFIRM, will determine the effectiveness of MDV3100 in patientswho have previously failed chemotherapy treatment with docetaxel. InNovember 2011, this trial was halted after an interim analysis revealedthat patients given the drug lived for approximately 5 months longerthan those taking placebo. As noted above, these preliminary reportswere reported to the public in early 2012. Medivation is expected tofile for FDA approval sometime in 2012.

There is another phase III trial, known as PREVAIL, that isinvestigating the effectiveness of MDV3100 with patients who have notyet received chemotherapy. As of October 2011, this trial is still opento accrual. In addition, a phase II trial began in March 2011 comparingMDV3100 with a commonly used anti-androgen, bicalutamide, in prostatecancer patients who have progressed while on LHRH analogue therapy(e.g., leuprorelin) or surgical castration.

III. Prostate Cancer Therapy

Classically, metastatic prostate cancer is treated with first lineandrogen deprivation therapy (ADT). ADT lowers serum testosterone, thehormone that drives proliferation and progression of the disease throughactivation of the androgen receptor (AR) within the cancer cell.Patients respond to ADT for a finite duration, on average 2-3 years,after which, they are considered to have failed first line hormonaltherapy and are considered to have castration resistant prostate cancer(CRPC). CRPC is the lethal form of the disease, causing over 30,000deaths in the United States alone.

The decapeptide gonadotropin-releasing hormone, also referred to asLHRH, was isolated in 1971. Chronic exposure to LHRH ultimatelysuppressed testosterone by desensitizing pituitary cells throughdownregulation of the LHRH receptors. Substitutions at the sixth aminoacid position of LHRH resulted in significantly more potent LHRHagonists. The monthly depot of leuprolide was the first LHRH agonistevaluated as a treatment for advanced prostate cancer. In a randomizedclinical trial, leuprolide was equivalent to 3 mg of DES in reducingserum testosterone to castrate levels. The advantage of leuprolide was alower incidence of cardiovascular toxicity. Leuprolide ultimatelyreplaced DES and orchiectomy as the preferred approach to androgendeprivation.

Over the next 30 years, substitutions at the sixth amino acid positionyielded goserelin, triptorelin, and histrelin, which are thecommercially available LHRH agonists in the United States. LHRH agonistsare differentiated by their route of administration (intramuscularinjection vs subcutaneous injection vs subcutaneous implant) andfrequency of administration (1-12 months). All of these LHRH agonistsseem to have similar side effect profiles and the ability to lower serumtestosterone to castrate levels. There has only been 1 study directlycomparing different LHRH agonists. Overall survival was significantlygreater with triptorelin compared with leuprolide, 97% vs 90.5% survivalat 9 months, respectively (P=0.033). Although not statisticallysignificant, there was a trend for triptorelin to better maintaincastrate levels of testosterone over a 9-month interval.

One of the paradigm changing discoveries in the management of CRPC isthat despite having progressed despite ADT, CRPC remains in large partdriven by active androgen receptor signaling. This has led to thedevelopment of therapies targeting the androgen axis includingabiraterone (De Bono et al., 2011), which decreases the production ofother ligands similar in function to testosterone, and most recentlyMDV3100, a second generation, potent inhibitor of the androgen receptor.Although MDV3100 improves survival, its benefits are limited, with themedian duration of benefit (progression free survival in the pivotalphase III trial) of just over 8 months. After progression on MDV3100 themedian survival is under 1 year, and it is unclear how the prostatecancer becomes resistant to MDV3100 and similar potent AR inhibitors.

According to the present invention, prostate cancers resistant to ARinhibition can now be treated with GR antagonists. Thus, it isspecifically contemplated that one or more of the GR antagonistsdiscussed herein or in the incorporated references may be used in thetreatment of prostate cancer, and in particular, CRPC. It is alsocontemplated that in some embodiments, more than one GR antagonist isemployed, while in other embodiments, only one is employed as part ofthe therapeutic method (though it may be administered multiple times).It is contemplated that the second one may be administered concurrentlywith the first one or they may be administered at different times.

Moreover, GR antagonist therapies may be combined, advantageously, withconventional cancer therapies. These include one or more selected fromthe group of chemical or radiation based treatments and surgery.Chemotherapies include, for example, cisplatin (CDDP), carboplatin,cabazitaxel, mitoxantrone, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen,raloxifene, estrogen receptor binding agents, taxol, gemcitabine,navelbine, farnesyl-protein transferase inhibitors, transplatinum,5-fluorouracil, vincristin, vinblastin and methotrexate, or any analogor derivative variant of the foregoing.

Suitable therapeutic agents include, for example, vinca alkaloids,agents that disrupt microtubule formation (such as colchicines and itsderivatives), anti-angiogenic agents, therapeutic antibodies, EGFRtargeting agents, tyrosine kinase targeting agent (such as tyrosinekinase inhibitors), serine kinase targeting agents, transitional metalcomplexes, proteasome inhibitors, antimetabolites (such as nucleosideanalogs), alkylating agents, platinum-based agents, anthracyclineantibiotics, topoisomerase inhibitors, macrolides, therapeuticantibodies, retinoids (such as all-trans retinoic acids or a derivativesthereof); geldanamycin or a derivative thereof (such as 17-AAG), andother standard chemotherapeutic agents well recognized in the art.

Certain chemotherapeutics are well known for use against prostatecancer. These prostate cancer chemotherapeutics are capecitabine,carboplatin, cyclophosphamide (Cytoxan), cabazitaxel, daunorubicin,docetaxel (Taxotere), doxorubicin (Adriamycin), epirubicin (Ellence),fluorouracil (also called 5-fluorouracil or 5-FU), gemcitabine,eribulin, ixabepilone, methotrexate, mitomycin C, mitoxantrone,paclitaxel (Taxol), thiotepa, vincristine, vinorelbine.

In some embodiments, the chemotherapeutic agent is any of (and in someembodiments selected from the group consisting of) adriamycin,colchicine, cyclophosphamide, actinomycin, bleomycin, daunorubicin,doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone,fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin,etoposide, interferons, camptothecin and derivatives thereof,phenesterine, taxanes and derivatives thereof (e.g., paclitaxel andderivatives thereof, taxotere and derivatives thereof, and the like),topetecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404,nab-5800, nab-5801, Irinotecan, HKP, Ortataxel, gemcitabine, Herceptin®,vinorelbine, Doxil®, capecitabine, Gleevec®, Alimta®, Avastin®,Velcade®, Tarceva®, Neulasta®, Lapatinib, STI-571, ZD1839, Iressa®(gefitinib), SH268, genistein, CEP2563, SU6668, SU11248, EMD121974, andSorafenib.

In some embodiments, the chemotherapeutic agent is a compositioncomprising nanoparticles comprising a thiocolchicine derivative and acarrier protein (such as albumin).

In further embodiments, a combination of chemotherapeutic agents isadministered to prostate cancer cells. The chemotherapeutic agents maybe administered serially (within minutes, hours, or days of each other)or in parallel; they also may be administered to the patient in apre-mixed single composition. The composition may or may not contain aglucocorticoid receptor antagonist. Combinations of prostate cancertherapeutics include, but are not limited to the following: AT(Adriamycin and Taxotere), AC±T: (Adriamycin and Cytoxan, with orwithout Taxol or Taxotere), CMF (Cytoxan, methotrexate, andfluorouracil), CEF (Cytoxan, Ellence, and fluorouracil), FAC(fluorouracil, Adriamycin, and Cytoxan), CAF (Cytoxan, Adriamycin, andfluorouracil) (the FAC and CAF regimens use the same medicines but usedifferent doses and frequencies), TAC (Taxotere, Adriamycin, andCytoxan), and GET (Gemzar, Ellence, and Taxol).

The term “a serine/threonine kinase inhibitor,” as used herein, relatesto a compound which inhibits serine/threonine kinases. An example of atarget of a serine/threonine kinase inhibitor includes, but is notlimited to, dsRNA-dependent protein kinase (PKR). Examples of indirecttargets of a serine/threonine kinase inhibitor include, but are notlimited to, MCP-1, NF-κB, eIF2a, COX2, RANTES, IL8, CYP2A5, IGF-1,CYP2B1, CYP2B2, CYP2H1, ALAS-1, HIF-1, erythropoietin and/or CYP1A1. Anexample of a serine/theronine kinase inhibitor includes, but is notlimited to, Sorafenib and 2-aminopurine, also known as1H-purin-2-amine(9CI). Sorafenib is marketed as NEXAVAR. The compoundscan be used in combination with a glucocorticoid receptor antagonist.

The term “an angiogenesis inhibitor,” as used herein, relates to acompound which targets, decreases or inhibits the production of newblood vessels. Targets of an angiogenesis inhibitor include, but are notlimited to, methionine aminopeptidase-2 (MetAP-2), macrophageinflammatory protein-1 (MIP-1α), CCLS, TGF-β, lipoxygenase,cyclooxygenase, and topoisomerase. Indirect targets of an angiogenesisinhibitor include, but are not limited to, p21, p53, CDK2 and collagensynthesis. Examples of an angiogenesis inhibitor include, but are notlimited to, Fumagillin, which is known as 2,4,6,8-decatetraenedioicacid, mono[3R,4S,5 S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3 -(3-methyl-2-butenyl)oxi-ranyl]-1-oxaspiro[2.5]oct-6-yl]ester,(2E,4E,6E,8E)-(9CI); Shikonin, which is also known as1,4-naphthalenedione,5,8-dihydroxy-2-[(1R)-1-hydroxy-4-methyl-3-pentenyl]-(9CI); Tranilast,which is also known as benzoic acid,2-[[3-(3,4-dimethoxyphenyl)-1-oxo-2-propenyl]amino]-(9CI); ursolic acid;suramin; thalidomide and lenalidomide, and marketed as REVLIMID. Thecompounds can be used in combination with a glucocorticoid receptorantagonist.

Radiation therapies that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors affect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells. In particular, intravenousadministration of samarium, strontium and radium-223 are contemplated.

Laser therapy is the use of high-intensity light to destroy tumor cells.Laser therapy affects the cells only in the treated area. Laser therapymay be used to destroy cancerous tissue and relieve a blockage in theesophagus when the cancer cannot be removed by surgery. The relief of ablockage can help to reduce symptoms, especially swallowing problems.Photodynamic therapy (PDT), a type of laser therapy, involves the use ofdrugs that are absorbed by cancer cells; when exposed to a speciallight, the drugs become active and destroy the cancer cells. PDT may beused to relieve symptoms of esophageal cancer such as difficultyswallowing.

Immunotherapeutics, generally, rely on the use of immune effector cellsand molecules to target and destroy cancer cells. The immune effectormay be, for example, an antibody specific for some marker on the surfaceof a tumor cell. The antibody alone may serve as an effector of therapyor it may recruit other cells to actually affect cell killing. Theantibody also may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a tumor cell target. Various effector cells includecytotoxic T cells and NK cells.

Gene therapy is the insertion of polynucleotides, including DNA or RNA,into an individual's cells and tissues to treat a disease. Antisensetherapy is also a form of gene therapy in the present invention. Atherapeutic polynucleotide may be administered before, after, or at thesame time of a first cancer therapy. Delivery of a vector encoding avariety of proteins is encompassed within the invention. For example,cellular expression of the exogenous tumor suppressor oncogenes wouldexert their function to inhibit excessive cellular proliferation, suchas p53, p16, FHIT and C-CAM.

Additional agents to be used to improve the therapeutic efficacy oftreatment include immunomodulatory agents, agents that affect theupregulation of cell surface receptors and GAP junctions, cytostatic anddifferentiation agents, inhibitors of cell adhesion, or agents thatincrease the sensitivity of the hyperproliferative cells to apoptoticinducers. Immunomodulatory agents include tumor necrosis factor;interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K andother cytokine analogs; or MIP-1, MIP-1β, MCP-1, RANTES, and otherchemokines. It is further contemplated that the upregulation of cellsurface receptors or their ligands such as Fas/Fas ligand, DR4 orDR5/TRAIL would potentiate the apoptotic inducing abilities of thepresent invention by establishment of an autocrine or paracrine effecton hyperproliferative cells. Increases intercellular signaling byelevating the number of GAP junctions would increase theanti-hyperproliferative effects on the neighboring hyperproliferativecell population. In other embodiments, cytostatic or differentiationagents can be used in combination with the present invention to improvethe anti-hyperproliferative efficacy of the treatments. Inhibitors ofcell adhesion are contemplated to improve the efficacy of the presentinvention. Examples of cell adhesion inhibitors are focal adhesionkinase (FAKs) inhibitors and Lovastatin. It is further contemplated thatother agents that increase the sensitivity of a hyperproliferative cellto apoptosis, such as the antibody c225, could be used in combinationwith the present invention to improve the treatment efficacy.

Various combinations with a glucocorticoid receptor antagonist and ananticancer agent or compound (or a combination of such agents and/orcompounds) may be employed, for example glucocorticoid receptorantagonist is “A” and the conventional anticancer therapy or agent (or acombination of such agents) given as part of an anticancer therapyregime, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/A/B B/A/B/A A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the therapy or agents to a patient will follow generalprotocols for the treatment/administration of such compounds, takinginto account the toxicity, if any, of the therapy. It is expected thatthe treatment cycles would be repeated as necessary. It also iscontemplated that various standard therapies, as well as surgicalintervention, may be applied in combination with the described therapy.

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a GR antagonist and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery is a cancer treatment that may beused in conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies.

Curative surgery includes resection in which all or part of canceroustissue is physically removed, excised, and/or destroyed. Tumor resectionrefers to physical removal of at least part of a tumor. In addition totumor resection, treatment by surgery includes laser surgery,cryosurgery, electrosurgery, and microscopically controlled surgery(Mohs' surgery). It is further contemplated that the present inventionmay be used in conjunction with removal of superficial cancers,precancers, or incidental amounts of normal tissue. Upon excision ofpart of all of cancerous cells, tissue, or tumor, a cavity may be formedin the body. Treatment may be accomplished by perfusion, directinjection or local application of the area with or without an additionalanti-cancer therapy.

Other aspects of therapy include assessing AR or GR expression oractivity. Methods for assessing the level of expression or activity ofthese receptors is described in greater detail in U.S. PatentPublication No. 2011/0269728, the entire contents of which are herebyincorporated by reference.

IV. Pharmaceutical Formulations, Routes of Administration and Kits

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions in a form appropriate for theintended application. Generally, this will entail preparing compositionsthat are essentially free of pyrogens, as well as other impurities thatcould be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. Such routes includeoral, nasal, buccal, rectal, vaginal or topical route. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal, or intravenous injection. Suchcompositions would normally be administered as pharmaceuticallyacceptable compositions, described supra. Of particular interest isdirect intratumoral administration, perfusion of a tumor, oradministration local or regional to a tumor, for example, in the localor regional vasculature or lymphatic system, to tumor vasculature or ina resected tumor bed.

Treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, or 12 months. These treatments may be of varying dosages aswell. A patient may be administered a single compound or a combinationof GR antagonist described herein in an amount that is, is at least, oris at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, or 100 mg/kg (or any range derivable therein). A patient maybe administered a single GR antagonist or a combination of compoundsdescribed herein in an amount that is, is at least, or is at most 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380,390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500mg/kg/day (or any range derivable therein).

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the polypeptides of the present invention may beincorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientmay also be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences,” 15^(th) Ed., 1990). Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Kits may provided with one or more agents according to the presentinvention, for example, in the form of a container with a label.Suitable containers include, for example, bottles, vials, and testtubes. The containers may be formed from a variety of materials such asglass or plastic. The label on the container may indicate that thecomposition is used for a specific prognostic or therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above. The kit of the invention willtypically comprise the container described above and one or more othercontainers comprising materials desirable from a commercial and userstandpoint, including buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

V. Examples

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. The present examples, along with themethods described herein are presently representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

EXAMPLE 1 Materials and Methods

Human Tissue Procurement. Tissue microarrays were constructed by theHuman Tissue Research Center at the University of Chicago Medical Centerwith Institutional Review Board approval. As described previously, anested case control was used to select 138 prostate cancer samples fromover 500 consecutive radical prostatectomies performed at the Universityof Chicago between 1995 and 2002 for the tissue microarray used in thisstudy (Lotan et al., 2007). Areas involved by prostate cancer andadjacent non-neoplastic prostatic tissue were punched (2 mm cores) fromthe formalin-fixed, paraffin embedded samples and arrayed with 72-108cores per slide. These tissues are referred to as “treatment-naiveprostate cancer” (TN-PC). Of this total (excluding duplicates), 126samples had a reported Gleason grade, 125 had available staging data,and 122 had an associated preoperative PSA. In addition, 18 full tissuesections of prostate cancer following hormonal treatment (androgendeprivation±anti-androgen or anti-androgen receptor therapy) were cutinto 4-μm sections, mounted on slides for immunohistochemistry staining.These samples are referred to as “androgen-deprived prostate cancer”(AD-PC). These 18 samples included prostate cancer tissue samples from10 radical prostatectomies after preoperative androgen deprivation, 6transurethral prostatectomies, 1 pelvic exenteration, and 1 femoral headbiopsy with metastatic prostate cancer. Five of these 18 full sectionswere samples from bona fide “castrate-resistant” prostate cancers inthat the samples were from patients with progression of disease despitepharmacologic or surgical castration. Patient age ranged between 42 and93 years (42-81 for treatment-naive and 48-93 for androgen-deprivedpatients), with a median age of 63 years, and a mean age of 62.4 years.Patient disease characteristics including grade, stage and PSA aredetailed in Table I. The majority of both treatment-naive andandrogen-deprived samples had a Gleason grade between 6 and 7 (75% and67%, respectively). The majority of prostatectomy samples wereorgan-confined as pathologic stage T2 (56%). Twenty-eight patients (20%)had extra-capsular extension, and another 23 patients (17%) were notorgan-confined because of seminal vesicle invasion, invasion ofbladder/rectum, nodal involvement, or bone metastasis at the time ofsurgery.

TABLE 1 Prostate Cancer Tumor Charactertics Treatment-naiveAndrogen-deprived Gleason grade number (%) 5 23 (18.2) 3 (16.7) 6 35(27.8) 3 (16.7) 7 60 (47.6)   9 (50) 8-9  8 (6.4) 3 (16.7) Pathologicstage number (%) T2aN0  7 (5.6) T2bN0  11 (8.8) T2cN0 58 (46.4) T2cN1  1(0.8) T3aN0 27 (21.6) T3aN1  3 (2.4) T3bN0  12 (9.6) T3bN1  3 (2.4) T4 3 (2.4) PSA Mean (± standard deviation) 9.0 (7.35)  Median (range) 6.75(3.3-43.8)

Immunohistochemistry. Immunohistochemical stains for androgen (AR) andGR as well as SGK1 were performed as previously described (Sahoo et al.,2005; Niemeier et al., 2010; Belova et al., 2009). Briefly, slides werecovered with xylene for a total of 10 min, then immersed in ethanol ofdecreasing dilutions (100% ethanol to 70% ethanol) and washed withTris-buffered saline (TBST) for 2 min. Antigens were unmasked withsodium citrate buffer pH 6.0 followed by heat treatment at 95° C. Aftercooling, slides were placed in 3% hydrogen peroxide for 20 min and thenwashed in TBST. Next, to block endogenous peroxidase and protein, slideswere incubated in the dark in a milk-peroxide solution (90 parts dH₂O, 5parts skim milk, and 5 parts 3% hydrogen peroxide) and then again washedin TBST. The primary antibody, in blocking buffer, was then applied tothe slide. The slides were incubated with the primary antibody for 30min and then washed. After application of the primary antibody, the DAKOEnVision+ System-HRP (DAB (3,3′-diaminobenzidine)) was used. Omittingthe primary antibody step served as a negative control for all tissues.The following antibodies were used: anti-AR (AR441 1:50, Dako), anti-GR(NCLGCR 1:20, Novocastra), and anti-SGK1 (1:150, Affinity Bioreagents, CTerminal SGK1 antibody). All antibodies were assessedsemi-quantitatively in both prostate cancer and adjacent non-neoplasticprostatic tissue. Any nuclear and/or cytoplasmic immunoreactivity wasrecorded. Intensity was graded on a four-point scale from 0 to 3 torepresent negative, weak, moderate, and strong, respectively. Thisscoring was performed by two pathologists who reviewed theimmunoreactivity together and arrived at a consensus score that was usedin the analyses.

Follow-Up and Cancer Recurrence Analysis. Follow-up data were collectedusing the University of Chicago Prostate Cancer Database (UCPC). Allpatients undergoing surgery, in this case radical prostatectomy, arecaptured in the electronic UCPC Database. The database contains detaileddemographic, surgical, pathological, functional as well as long-termoncologic outcome data. The follow-up and outcome data were collectedusing surveys (mail, e-mail, and phone) at, 3, 6, 12, and 24 monthsafter surgery and annually thereafter. Professional support by theSurvey Lab at University of Chicago was utilized. Oncologic outcome wasconfirmed by mailed/emailed/faxed tumor marker (PSA) results, reportsfor adjuvant treatments as well as queries to death registries. Forprostate cancer recurrence analysis, PSA recurrence post-prostatectomywas defined as any PSA value above the lower limits of detection.Patients whose PSA failed to nadir to undetectable post-prostatectomywere considered to have met the recurrence endpoint at the date of firstPSA follow-up. Patients were censored from the analysis at the time oflast follow-up if they were alive and had not met criteria for PSArecurrence. Time to PSA progression was calculated for groups based onGleason grade and stage. Gleason scores 5 and 6 were considered lowgrade and were combined for this analysis. Similarly the Gleason scores7, 8, and 9 were grouped as they represent more aggressive biology.Although it has been reported that Gleason sum 4+3 versus 3+4 cancersbehave differently, many of the pathology reports available from thistissue set did not differentiate between the two, but rather listed thecancer as “Gleason 7.” Thus, the group was analyzed together (Chan etal., 2000; Wright et al., 2009).

Statistical Analysis. Data were summarized using descriptive statistics.Fisher's exact test was used to compare categorical variables (e.g.,high vs. low staining levels) between groups. The Kaplan and Meiermethod was used to estimate progression-free survival (PFS). Univariateanalyses comparing PFS between groups were performed using the log-ranktest. In addition, the Wilcoxon-Breslow-Gehan log-rank test, which giveshigher weight to the earlier data points, was used to compare PFSbetween SGK-1 expression groups. Univariate Cox proportional hazardsregression models were used to estimate hazard ratios between groups.PFS rates at 2 and 5 years were compared using a chi-squared test with acomplementary log-log transformation of the Kaplan-Meier estimator(Klein et al., 2007).

GR Antagonist Studies. CWR-22RV1 (22RV1) and LAPC4 prostate cancer celllines were grown in vitro with various combinations of the AR antagonistMDV3100 (10 μM), synthetic androgen R1881 (10 nM), GR agonistdexamethasone (Dex, 1 μM or 100 nM, the GR inhibitor mifepristone (Mif,100 nM), or the SGK1 kinase inhibitor GSK650394 (1 μM). SGK1-FLAG orvector was stably expressed and cellular viability, secreted PSA, andprotein lysates were collected.

EXAMPLE 2

Immunohistochemistry of SGK1, GR, and AR Expression. AR and SGK1 wereexpressed in essentially all prostate epithelium including TN-PC, AD-PC,and unaffected adjacent prostate tissue (UPT). As expected, nuclear ARexpression was uniformly strong (3+) in all prostate cancer tissuesamples regardless of treatment status (Table II, FIGS. 1A-C). SGK1expression was consistently more intense in the prostate cancer samplescompared to UPT (FIGS. 1A-C). Additionally, SGK1 expression was moreintense in the nucleus versus the cytoplasm. As SGK1 expression byimmunohistochemistry was strong in the majority of specimens, thefour-point scale for SGK1 expression was grouped as high (3+) versus low(0, 1, and 2+) for further analysis. With this classification, 100 of126 (79%) TN-PC samples were strongly positive for SGK1 compared to only8 out of 18 (44%) AD-PC samples (P ¼ 0.003) (Table II, FIGS. 1A-C).There was no association between SGK1 expression and tumor stage. Thepercentage of patients with a low Gleason score (Gleason sum 5 or 6) andlow SGK1 expression was nearly half of that compared to the percentageof high Gleason grade (7-9) tumors with low SGK1 expression (13.8% vs.26.5%, P ¼ 0.08). This implies that SGK1 expression may inverselycorrelate with prostate tumor grade.

TABLE 2 SGK1 3+ (%)* AR+ (%) GR+ (%)** (n = 126) (n = 126) (n = 126)TN-PC 100 (79) 126 (100) 48 (38) AD-PC  8 (44)  18 (100) 14 (78) *P =0.003, **P = 0.002 (Fisher's exact test).

The inventors next examined GR expression. Unlike AR, which wasuniversally strongly positive regardless of whether the patient wasuntreated or androgen-deprived, GR expression was more variable.Specifically, GR expression was present in a significantly higherproportion of AD-PC compared to TN-PC (78% vs. 38% positive staining, P¼ 0.002) (Table II, FIGS. 1A-C). Interestingly GR was highly expressedin four of five castrate-resistant AD-PC samples (80%), suggesting thatincreased GR activity may contribute to castration-resistance bybypassing AR blockade.

SGK1 Expression and Prostate Cancer Recurrence. To date, of the 122TN-PC patients for whom there is follow-up data available, PSArecurrence post-prostatectomy has occurred in 37.7% (n=46). Of the 76patients who have not met PSA recurrence criteria, the median follow-upis 5.5 years (range 0.11-15.4 years). The median progression-freesurvival [(PFS)=time from prostatectomy to PSA recurrence] for theentire cohort is 11.13 years (95% CI: 8.8-not reached) (FIG. 2A). Timeto PSA progression was also calculated for patient subgroups based ontumor Gleason grade and stage. As expected, there was a statisticallysignificant difference in PFS between the “low” and “high” Gleason gradesubgroups (P=0.004) (FIG. 2B), and patients with high Gleason scoreswere at a higher risk of progression (HR=2.51) on univariate analysis.Higher tumor stage was similarly associated with an increased risk ofrelapse (P<0.0001). Overall, there was a non-statistically significanttrend towards decreased PFS (worse outcome) associated with low (0 to2+) SGK1 expression compared to high SGK1 expression (log-rank P=0.116;Wilcoxon-Gehan-Breslow log-rank test P=0.077) (FIG. 2C). At 2 years,86.2% (95% CI: 77.3-91.7%) of patients were alive without progression inthe high SGK1 group versus only 71.4% (95% CI: 49.1-85.2%) alive withoutprogression in the low SGK1-expression group (P=0.083). At 5 years offollow-up, the percentage of patients without progression in the highSGK1 group was 72.6% (95% CI: 61.8-80.8%) versus only 47.8% (24.1-68.2%)in the low SGK1 group (P=0.034). These data support the hypothesis thatlow SGK1 expression in primary prostate tumors is associated with aworse clinical outcome compared to high SGK1 expression. This studyprovides strong supporting translational evidence from patient-derivedprostate tissue that AR signaling regulates SGK1 expression in prostatecancer. Interestingly, there is a suggestion that SGK1 expression isinversely associated with grade and cancer recurrence. It is possiblethat aberrant AR signaling is associated with a poorly differentiatedphenotype and unfavorable outcome. Following androgen deprivation, GRexpression increases and SGK1 expression remains high in a subset ofpatients. Further study is needed to clarify the role of the AR/GR/SGK1network in castration resistance. This study highlights an interestinginteraction between AR signaling, SGK1, and GR in prostate cancer.

GR Antagonist Studies. Sustained androgen receptor (AR) signaling isrequired for the development of castrate resistant prostate cancer(CRPC). As shown above, following androgen deprivation therapy forprostate cancer, tumor glucocorticoid receptor (GR) expression isincreased. The GR and AR are known to share similar DNA bindingsequences and to regulate several common target genes, including theanti-apoptotic protein serum/glucocorticoid regulated kinase 1 (SGK1).The inventors hypothesized that increased GR expression might compensatefor diminished AR signaling, thereby facilitating resistance to potentinhibitors of the AR through sustained expression of apoptosisinhibitor. Following MDV3100 treatment, GR expression increased in bothCWR-22RV1 and LAPC4 cell lines. Although proliferation rates of bothSGK1 and vector expressing cell lines were slower following Dex, therewas a relative protection from MDV3100-associated cell death. For LAPC4cells, cell survival compared to R1881 control was improved with Dex(66% vs. 47%, p<0.05). For 22RV1, Dex treatment also increased cellsurvival despite MDV3100 (151% vs. 51% without Dex). Dex treatment alsoled to increased PSA secretion despite MDV3100 treatment. For LAPC4cells, secreted PSA levels (ng/nL) were 22.7 (R1881), 1.4 (MDV3100), and31.5 (MDV3100+Dex). For the CWR-22RV1's there was similar antagonism ofMDV3100 activity following Dex treatment: 10.9, 4.4, and 41.9 for R1881,MDV3100 and MDV3100+Dex respectively. Expression of SGK1 was reducedwith MDV3100, but increased with the addition of Dex. When mif was addedin addition to MDV3100 treatment, there was a synergistic impairment ofcell survival. For LAPC4 cells, compared to R1881+Dex control, there wasno change in survival with the addition of Mif alone, but a significant26% and 52% reduction in cell death with MDV3100 and MDV3100+Mifrespectively.

To test if SGK1 was required for GR-mediated protection from cell deathfollowing AR inhibition, the cell lines were grown with or withoutGSK650394. Cell survival with both cell lines was impaired followingtreatment with the SGK1 inhibitor. For LAPC4, relative to R1881+Dexcontrol, % survival was 58% with MDV3100+Dex and only 19% with theaddition of GSK650394 (p<0.05). Results with the 22RV1 cells weresimilar. SGK1-Flag over-expressing LAPC4 cells were protected fromMDV3100 treatment compared to vector controls—61% vs. 24% survival(p<0.05). These data suggest that increased GR expression and activityantagonizes AR inhibition and sustains tumor cell survival. Furthermore,SGK1 appears to be a downstream mediator of this effect.

In conclusion, the inventors have shown that in the context of firstline ADT, the expression of glucocorticoid receptor (GR) within humanprostate cancer specimen increases compared to those who have not beentreated with ADT. With the understanding that CRPC is driven by ARsignaling, and that second generation AR inhibitors such as MDV3100potently inhibit the AR in this setting, the inventors set out to testif GR activation could potentially compensate for reduced AR function inthe setting of CRPC treated with MDV3100 thereby leading to resistanceto the anti-AR therapy. The preclinical data to date support the notionthat increased GR activity in CRPC treated with AR inhibitors such asMDV3100 can compensate for diminished AR activity and can protect CRPCcells from MDV3100 (and other AR inhibitor)-associated cell death. Inthe inventors' preclinical models with CRPC cell lines, the addition ofMDV3100 significantly increases GR expression level within 3-7 days.Furthermore, with the addition of a GR inhibitor, such as mifepristone,the GR activity can be overcome, restoring the decrease in proliferationseen with MDV3100 in the absence of GR activation. Clinically, this hashigh-level potential implications. CRPC patients have endogenouscirculating and local hormones (glucocorticoids) that can activate theGR at baseline. Furthermore, CRPC patients are often treated with higherdoses of synthetic glucocorticoids used for their anti-inflammatoryproperties. Thus, CRPC patients who are treated with MDV3100 may haveactivated GR within their prostate cancers that could drive resistanceto AR inhibition based on the data described above. Thus, the inventorsbelieve that a GR inhibitor will synergize with second generationAR-inhibitors, such as MDV3100, and delay the onset of CRPC progressionin patients treated with the combination compared to patients treatedwith MDV3100 alone.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods, and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

IV. References

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of treating prostate cancer in a subjectreceiving anti-androgen receptor (anti-AR) therapy, said prostate cancerhaving become resistant to said anti-AR therapy, the method comprisingadministering a glucocorticoid receptor (GR) antagonist in addition tothe anti-AR therapy.
 2. The method of claim 1, wherein said subject haspreviously been or is being treated with androgen deprivation therapy.3. The method of claim 2, wherein said androgen deprivation therapycomprises treatment with leuprolide, goserelin, triptorelin, histrelin,degerelix or surgical castration.
 4. The method of claim 1, wherein saidsubject has been or is being treated with an androgen receptor (AR)antagonist.
 5. The method of claim 4, wherein the AR antagonist isMDV3100, ARN-509, flutamide, bicalutamide, nilutamide, or cyproteroneacetate.
 6. The method of claim 1, wherein said prostate cancer in saidsubject exhibits elevated GR expression.
 7. The method of claim 1,wherein said GR antagonist is beclometasone, betamethasone, budesonide,ciclesonide, flunisolide, fluticasone, GSK650394, mifepristone,mometasone, or triamcinoclone.
 8. The method of claim 1, wherein saidsubject is treated with a second prostate cancer therapy.
 9. The methodof claim 8, wherein said second prostate cancer therapy is conventionalchemotherapy, radiotherapy, cryotherapy, immunotherapy or surgery. 10.The method of claim 8, wherein the second prostate cancer therapy isgiven prior to, or at the same time, as said GR antagonistadministration.
 11. The method of claim 8, wherein the second prostatecancer therapy is given after said GR antagonist administration.
 12. Amethod of treating prostate cancer in a subject receiving anti-androgenreceptor (anti-AR) therapy, said prostate cancer having become resistantto anti-AR therapy, the method comprising co-administering to saidsubject a glucocorticoid receptor (GR) antagonist and a cytotoxicchemotherapy.
 13. The method of claim 12, wherein said subject haspreviously been or is being treated with androgen deprivation therapy.14. The method of claim 13, wherein said androgen deprivation therapycomprises treatment with leuprolide, goserelin, triptorelin, histrelin,degerelix or surgical castration.
 15. The method of claim 12, whereinsaid subject has been or is being treated with an androgen receptor (AR)antagonist.
 16. The method of claim 15, wherein the AR antagonist isMDV3100, ARN-509, flutamide, bicalutamide, nilutamide, or cyproteroneacetate.
 17. The method of claim 12, wherein said prostate cancer insaid subject exhibits elevated GR expression.
 18. The method of claim12, wherein said GR antagonist is beclotnetasone, betamethasone,budesonide, ciclesonide, flunisolide, fluticasone, GSK650394,mifepristone, mometasone, or triamcinoclone.
 19. The method of claim 12,wherein said subject is treated with a second prostate cancer therapy.20. The method of claim 19, wherein said second prostate cancer therapyis conventional chemotherapy, radiotherapy, cryotherapy, immunotherapyor surgery.
 21. The method of claim 19, wherein the second prostatecancer therapy is given prior to, or at the same time, as said GRantagonist.
 22. The method of claim 19, wherein the second prostatecancer therapy is given after said GR antagonist.